This application addresses broad Challenge Area (03): Biomarker Discovery and Validation, and specific Challenge Topic 03-DK-103: Identify the normal and diseased proteome of subcellular organelles of relevance to NIDDK diseases. Mitochondrial dysfunction has been postulated as a unifying theme of type 2 diabetes mellitus (T2DM), which currently affects more than 20 million individuals in the US alone. Decreased mitochondrial content and oxidative capacity, altered fatty acid oxidation, and a rise in mitochondria-derived reactive oxygen species have been associated with the development of insulin resistance and T2DM. However, the importance of these observations has recently been challenged, and it remains unclear which, if any, changes to mitochondrial composition and/or function actually contribute to T2DM etiology. The long-term objective of this proposal is to arrive at a fuller understanding of the importance of mitochondrial dysfunction in T2DM by elucidating the compositional changes that accompany the onset of this disease at unprecedented resolution. Despite the observations noted above, an understanding of the molecular basis for mitochondrial dysfunction in T2DM has largely been lacking for at least two reasons. First, until recently, much of the mammalian mitochondria proteome itself was undefined, making it impossible to conduct comprehensive comparisons of mitochondrial composition during different stages of this disease. Second, microarray analyses used to investigate global differences in gene expression between healthy and diabetic patients have been crippled by the markedly poor correlation between cellular mRNA and protein levels. Here, we propose to establish a comprehensive map of the proteomic and phosphoproteomic changes that occur during the onset of obesity-dependent T2DM using state-of-the-art quantitative proteomics (AIM1). To do so, we will take advantage of two mouse strains: C57BL/6 (B6) leptinob/ob mice, which are resistant to diabetes, and BTBR leptinob/ob mice, which develop severe diabetes as they age. During the two-year timeframe of this project, we will focus on mitochondria from skeletal muscle, the primary site of insulin- mediated glucose disposal in the body. This proteomic resource will also provide an opportunity to explore the mechanisms by which these mitochondrial alterations occur. In particular, we will use integrative genomics to elucidate the post-transcriptional and post-translational mechanisms at play in the control of mitochondrial gene expression and protein function (AIM2). Completion of our aims will clarify the mitochondrial restructuring in skeletal muscle that accompanies the onset of T2DM, provide a rich quantitative proteomic resource for the diabetes community, and lay the groundwork for identifying protein biomarkers and designing muscle-specific therapies targeted against this organelle to treat T2DM. PUBLIC HEALTH RELEVANCE: Mitochondrial dysfunction is a prominent feature of type 2 diabetes mellitus (T2DM), but the underlying basis for this dysfunction is not well understood. We propose to use state-of-the-art proteomics technologies to establish a map of mitochondrial alterations that occur in skeletal muscle during the onset of obesity-induced diabetes. Completion of this goal will help identify mitochondrial biomarkers for this disease, and provide a framework for designing therapies targeted against this organelle to treat T2DM.